3.1 Endophytic and Rhizospheric fungal isolates
Altogether 89 and 38 fungal isolates were obtained from healthy plant tissues and rhizospheric soil respectively. By comparing colony and spore morphologies with standard identification manual (Barnett and Hunter 1998) it was found that the isolates were comprised of fungal genera Aspergillus, Colletotrichum, Fusarium, Penicillium, and non-sporulating mycelia sterilia. The colonization/isolation frequencies of endophytic and rhizospheric fungi is presented in Fig. 1
3.2 Principle Component Analysis (PCA):
The PCA showed that the genus Penicillium is dominant in leaves of litchi plants, whereas Mycelia sterilia is dominant over barks. Similarly, Fusarium was dominant on root tissues and Aspergillus was dominant in rhizospheric soil. In Fig 2 the PCA biplots is shown with active variables and active observations.
3.3 IAA production and phosphate solubilization
Out of 127 isolates tested for the production of IAA; 19 endophytic and 9 rhizospheric fungi demonstrated notable production of IAA. It has been observed numerously that there is a positive correlation between the production of IAA, phosphate solubilization and disease resistance in fungi (Bader et al. 2020; Napitupulu et al. 2021). Consequently, the phosphate solubilization activity of these (19+9=28) isolates was evaluated. With 143.136±3.94 µgml-1, the endophytic isolate BE23 showed the highest IAA production, followed by LE20 (118.058±1.98 µgml-1). Whereas, rhizospheric fungal isolate SF32 demonstrated an exceptionally elevated production of IAA, with a concentration of 179.25±3.75 µgml-1. Statistical analysis showed the 28 isolates were grouped in 17 groups which showed significant difference across groups by p≤ 0.05 in production of IAA in presence of tryptophan. However, 9 groups were formed among the isolates showing production of IAA showing significant difference. Thus, indicating that production of IAA is independent in presence of tryptophan among the isolates.
Phosphate solubilization was found to be positive in 12 isolates. The documented PSA values of 8 endophytic isolates were between 1.8 and 3.4 SI. Isolate LE07 showed the highest phosphate solubilization of 3.442±0.04SI and 4 rhizospheric fungal isolates exhibited PSA values between SI 1.9 and 3.1. Among them, isolate SF11 displayed the highest PSA, measuring SI 3.16±0.13. Table 1 displays the IAA production and PSA for each of the 28 isolates. Statistically, it was observed that the isolates had 7 groups having significant difference in PSA with p≤0.05.
3.4 Antagonistic activity
Strong inhibition of A. alternata was noted by the tested endophytic fungi (>55%). With 79±0.50% inhibition, the endophytic isolate LE24 showed the highest level of suppression, followed by BE14 with 78.5±0.61%. Similarly, rhizospheric isolate SF04 displayed the highest inhibition, at 80.8±0.50 followed by isolate SF32 with inhibition, at 80.5±0.54%. Table 2 shows the fungal isolates and their respective inhibition ability against A. alternata. Fig 3: displays the photo-plates of dual culture assay of LE24, BE14, SF32, and SF04 along with their graphical representations. Statistics showed the isolates had 8 related groups having significant difference across them with p≤0.05.
3.5 Antifungal Assay
The result from antifungal assay with crude metabolites of 4 promising isolates (LE24, BE14, SF32, and SF04) showed a zone of inhibition > 25mm. However, the isolate BE14 revealed exceptionally high antifungal activity with the zone of inhibition of 41.9±1.46 mm. Table 3 lists the results of antifungal activity of the 4 potent fungal isolates. Fig 4: displays the photoplates showing the zone of inhibitions.
3.6 Molecular identifications of the potent isolates
The isolates BE14 and SF32 were found to have the best antifungal activity and IAA production respectively. Formerly, BE14 morphologically identified as Pencillium sp. and SF32 as Aspergillus sp. were sequenced for 18s rDNA for ITS1 and ITS4 for their molecular identifications. The sequences were submitted to GenBank and the accession number was obtained as PP203028 and PP196577 respectively. The evolutionary history was done using Maximum Parsimony Method. The BLAST analysis revealed that BE14 (Penicillium sp.) had the closest homology with Penicillium citrinum and SF32 (Aspergillus sp.) with Aspergillus aculeatus (Table 4). 9 (Penicillium) and 8 (Aspergillus) sequences with the complete ITS rDNA region (18S rDNA-ITS1-5.8S-ITS2-rDNA) were taken into consideration for phylogenetic analysis after the sequences underwent filter search. The generated tree revealed that the isolate BE14 clustered with Penicillium citrinum and isolate SF32 clustered with Aspergillus aculeatus (Fig 5).
3.7 Characterization of crude metabolite
FTIR analysis of the crude metabolite of P. citrinum showed bands at 3438 cm-1, 2914 cm-1, 1417 cm-1, 1638 cm-1, 1310 cm-1, 1044 cm-1, 776 cm-1 and 698 cm-1. The bands were compared with standard ranges of functional groups given by (Lingegowda et al. 2012). Band at 3438 cm-1 corresponds to the stretching vibrations of alcohol, whereas, the band at 2914 cm-1 correspond to the stretching vibrations of alkanes. Similarly, the bands at 1417 cm-1 and 1638 cm-1 correspond to unsaturated aliphatic and Nitro compounds, bands at 1310 cm-1 to alkenes, bands at 1044 cm-1 to carbonyl and ether, bands at 776 cm-1 to nitrile, acyl, methoxy, and aromatic C-H groups and band at 698 cm-1 to alkyl halides. The FTIR chromatogram is shown in Fig 6 and the bands corresponding to each functional group are listed in Table 5
GC-MS analysis of P. citrinum metabolites extracted with ethyl acetate showed two major peaks viz. 35.560 min and 36.360 min having peak area of 13.012% and 9.892% respectively. The GC-MS chromatogram is shown in Fig 7. The probable volatile compounds for the first RT peak 35.560min were Succinic acid, 2,4,6-trichlorophenyl 3-methylbut-3-en-1-yl ester and 1,5-bis(3-Cyclopentylpropoxy)-1,1,3,3,5,5-hexamethyltrisiloxane. For the second peak, 36.360 min the two major volatile compounds were found to be Tris(tert butyldimethylsilyloxy)arsane and Hexamethylcyclotrisiloxane. The chemical formula, structure, etc. of these probable major compounds of ethyl acetate extracted crude metabolites have been presented in Table 6.